The locus coeruleus is directly implicated in L-DOPA-induced dyskinesia in parkinsonian rats: an electrophysiological and behavioural study

Abstract

Despite being the most effective treatment for Parkinson's disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.

Figure 1. Experimental design.

Experiment 1: At the beginning of the study, animals were lesioned with 6-OHDA injected into the right MFB. Two weeks later, the severity of the lesion was screened by forelimb use on a cylinder and amphetamine-induced rotations. To develop stable AIMs, rats were injected daily with 6 mg/kg L-DOPA (plus 12 mg/kg benserazide) or saline for 21 days, and AIMs were rated 2-3 days per week. At the end of the chronic treatment, L-DOPA was administered twice per week, and electrophysiological experiments were performed. Experiment 2: Animals were lesioned with 6-OHDA injected into the right MFB, and the severity of the lesion was screened in the 4^th week. DSP-4 (50 mg/kg, i.p.) was administered to some groups to induce an additional noradrenergic lesion. Between the 6^th and 9^th weeks, animals were treated daily with L-DOPA (6 mg/kg plus 12 mg/kg benserazide, s.c.) or saline, and AIMs were monitored 2-3 times per week. Experiment 3: Animals were lesioned, screened and AIMs were induced and evaluated. The severity of the AIMs was maintained by administering L-DOPA twice per week. On day 30 after the 6-OHDA lesion, dyskinetic animals were injected with ibotenic acid or vehicle into the right LC. Five days after noradrenergic damage, AIMs were evaluated regularly for four weeks. At the end of each study, all animals were transcardially perfused, and the brains were extracted, cut and stored for subsequent histological verification or immunohistochemical procedures.

Figure 2. Electrophysiological and histological verification of LC neuron recordings.

Example of raw trace of recorded action potentials (A). Scale-up of single spike from LC neuron recorded in vivo (B). Pictures were taken from Spike2 recordings. Histological verification of the recording site in the LC (C). Abbrev: LC, locus coeruleus; 4V, 4th ventricle; Me5, mesencephalic trigeminal nucleus.

Figure 3. Abnormal involuntary movements induced by chronic treatment with L-DOPA in 6-OHDA-lesioned animals.

Time course of changes in dyskinesia evaluated from the sum AIM scores of axial, limb and orolingual (A) or locomotive (B) during L-DOPA (6 mg/kg plus benserazide 12 mg/kg, s.c.) chronic treatment. Time course of sum AIM scores of axial, limb and orolingual (C) or locomotive (D) evaluated after a single injection of L-DOPA the last testing session (21^st day); n = 20, 13, 10 and 10 for 6-OHDA L-DOPA, 6-OHDA saline, sham L-DOPA and sham saline, respectively. Note that the sham L-DOPA, sham saline and 6-OHDA saline groups did not develop any abnormal movements.

Figure 4. Regression analysis of the severity of dyskinesia and the electrophysiological parameters of locus coeruleus neurons in dyskinetic animals.

The electrophysiological parameters of LC neurons are represented on the abscissa and the sum AIMs on the ordinate axis. The basal firing rate was correlated with the sum AIMs (A), and the axial (B), limb (C) and orolingual (D) subtypes. The coefficient of variation also correlated with the sum AIMs (F) and the axial (G), limb (H) and orolingual (I) subtypes. Locomotive behaviour was not correlated with the basal firing rate (E) or with the coefficient of variation in the LC (J). Each symbol represents an animal. Only 6-OHDA lesioned animals treated with L-DOPA (8 dyskinetic and 3 non-dyskinetic) are included in the analysis.

Figure 5. Abnormal movements induced by the chronic treatment with L-DOPA in animals lesioned with 6-OHDA or DSP-4+6-OHDA

. The time course of changes in dyskinesia evaluated from the sum (A), locomotive (B) or the global AIM score (C) induced by a 21-day treatment with L-DOPA (6 mg/kg plus benserazide 12 mg/kg, s.c.). Sum and locomotive AIM score was evaluated every testing session and global AIM score weekly. Time course of sum AIM scores of axial, limb and orolingual (D) or locomotive (E) evaluated after a single injection of L-DOPA the last testing session (21^st day). Only groups receiving L-DOPA are included in the graph (n = 10, 10, 6 and 5 for DSP₄+6-OHDA, 6-OHDA, DSP₄ and sham, respectively). Saline-treated animals, sham L-DOPA and DSP4 groups did not show any sign of dyskinesia. Tyrosine hydroxylase immunostaining in the striatum of sham and 6-OHDA-lesioned animals (F, upper boxes, scale bar 1 mm). Dopamine-β-hydroxylase immunostaining of sham and DSP-4-treated animals in the prefrontal cortex (F, bottom boxes, scale bar 200 µm). **p<0.01 vs 6-OHDA (one-way ANOVA followed by Bonferroni post-hoc test).

Figure 6. Involvement of the locus coeruleus in the development of abnormal involuntary movements induced by chronic treatment with L-DOPA.

Briefly, 6-OHDA-lesioned animals were injected daily with L-DOPA (6 mg/kg plus 12 mg/kg benserazide, s.c.) for 21 days and AIMs were evaluated twice a week. After a 21-day chronic treatment, L-DOPA was administered twice per week. On day 31, animals received an injection of ibotenic acid or vehicle into the right LC (solid line: LC-lesion group, n = 8; dash line: Sham group, n = 5), indicated in the graph with an arrow. Five days after, AIMs were evaluated for another 32 days in 10 additional testing sessions. A significant increase in sum AIM score (A) was observed in the LC-lesion group. Locomotive AIM remained unaltered after the LC lesion (B). In the LC-lesion group, time course of the L-DOPA-induced AIMs after a single injection of L-DOPA was significantly prolonged after the LC damage for the sum (C) but not for locomotive AIM score (D). Bar histograms representing separately axial (E), limb (F) and orolingual AIM scores (G). White or black bars are used for representing each session before or after the LC-lesion, respectively. Thionine-staining in the LC of 6-OHDA-lesioned animals with an additional LC-lesion (Hi). Dashed squares are magnified below, corresponding to the LC contralateral (Hii) and ipsilateral to the lesion (Hiii). All slices are 40 µm-thick and were stained using free-floating slice procedures. Scale bar: 400 µm (Hi) and 200 µm (Hii-Hiii). * p<0.05, ** p<0.01 and *** p<0.001 vs. sham group (repeated measures two way ANOVA followed by Bonferroni post-hoc test) # p<0.05, ## p<0.01 and ### p<0.001 vs. day 30th (repeated measures ANOVA followed by Dunnett’s post-hoc test) .